FRET microscopy is a very effective means to investigate protein:protein interactions in living cells. This project uses FRET microscopy to investigate the induced interactions between TCR and co-receptors that occur during antigen recognition. FRET microscopy will be used to analyze the different abilities of CD8a(3 and aa to act as coreceptors and to be recruited to the immunological synapse during antigen recognition, as well as to determine how endogenous peptides enhance T cell recognition, including enhancing the TCR:coreceptor interaction. The movement and interactions of Lck and Fyn during antigen recognition will be analyzed with fluorescent chimeras, and with a FRET biosensor. Differential sialylation of CDS has been found in developing thymocytes and in activated T cells. The role of sialylation in recruitment of CDS to the synapse, chain pairing, TCR:CD8 and CD8:Lck interactions will be investigated. Transgenic mice expressing CD3C-CFP and CD8(3-YFP will be used to image TCR:coreceptor interactions in cell conjugates between developing thymocytes and stromal cells, under different selecting conditions, to understand how the speed and intensity of the TCR:coreceptor interactions are modulated in positive and negative selection. These factors will also be analyzed for naive, effector and memory T cells. The interactions between the T-cell antigen receptor, coreceptors such as CD4,and signaling proteins, are crucial to the immune response. Understanding how these interactions take place will aid in improving the immune response to viruses or cancer, and in reducing autoimmune reactions.